Sequence-specific oligonucleotides containing modified internucleoside phosphodiester linkages have utility as antisense molecules for therapeutic applications and nucleic acid hybridization probes for diagnostic or therapeutic efficacy-monitoring applications.
Successful antisense molecules and nucleic acid hybridization probes must bind specifically to the single-stranded or double-stranded target nucleic acid sequence of interest under physiological conditions. Such molecules and probes must also be effectively taken up by intact cells and must be resistant to nuclease degradation.
The phosphodiester backbone has been modified in an attempt to satisfy these criteria. For example, the phosphodiester backbone has been replaced by phosphonate (Miller et al. (1980) J. Biol. Chem. 255:9659-9665), phosphotriester (Pless et al. (1977) Biochemistry 16:1239-1250) or phosphorothioate backbones (Stec et al. (1984) J. Am. Chem. Soc. 106:6077-6079).
One approach to oligonucleotide backbone modification has been to remove the negative charge of the internucleoside phosphodiester ("PDE") linkage to produce neutral backbones such as, for example, methyl phosphonates (Vyazovkina et al. (1994) Nucleic Acids Res. 22:2404-2409), phosphoramidates (Jager et al. (1988) Biochemistry 27:7237-7246) or peptide nucleic acids (Egholm et al. (1992) J. Am. Chem. Soc. 114:1895-1897).
An alternative approach to modifying the oligonucleotide backbone has been to replace anionic PDE groups with cationic groups. Cationic substituents have been attached to the internucleoside phosphorus atom via phosphoramidate linkages (Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470-4471). Cationic groups can also be introduced into oligonucleotides via phosphonate derivatives in which the anionic oxygen is replaced by a cationic group via a phosphorus-carbon bond. Thus, the preparation of oligonucleotides in which the backbone consists of alternating phosphodiester and stereoisomerically pure (2-aminoethyl)-phosphonate linkages, and oligonucleotide containing backbones consisting of (aminomethyl)-phosphonates have been respectively reported in Fathi et al. (1994) Nucleic Acids Res. 22:5416-5424 and Fathi et al. (1994) Bioconjugate Chem. 5:47-57.
Patil et al. (1994) Biorg. Medicinal Chem. Lett. 4:2663-2666 reported the synthesis of oligothymidylate analogs containing stereoisomers of phosphorothioates using stereoisomerically pure modified dinucleosides to synthesize decathymidylates with alternating stereoregulated anionic phosphorothioate and anionic PDE linkages.
Peyrottes et al. (1994) Nucleosides & Nucleotides 13:2135-2149 prepared oligomers with methoxyphosphoramidate internucleoside linkages using preformed dimers. Furthermore, when compared with oligo(dT), the oligomer with the methoxyphosphoramidate internucleoside linkages showed reduced thermal stability when hybridized to poly(dA) or poly(A).
There is a need in the art for oligonucleotides having cationic internucleoside linkages that have greater affinity target nucleic acids than oligonucleotides having exclusively standard phosphodiester internucleoside linkages. Such oligonucleotides can serve as antisense therapeutic agents and as probes in nucleic acid hybridization assays.